1. Field of the Invention
The present invention relates to a novel NAD synthetase, a process for production of said enzyme and an assay method using the same.
More particularly, the present invention relates to a novel NAD synthetase, which at least catalyzes the reaction (a) hereinbelow in the presence of Mg++ or Mn++ ion, and does not catalyze the reaction (b) hereinbelow in the presence of Mg++ ion, utilizes ammonia including ammonium ion as a substrate, and does not utilize at least glutamine and asparagine as a substrate, to a process for production of the same, and to an assay method of any one of ATP, deamide-NAD and ammonia including ammonium ion in a specimen by incubating the said specimen with the present NAD synthetase. ##STR3##
2. Description of the Prior Art
Heretofore, NAD synthetase has been known to exist in rat liver [J. Biol. Chem. 233, 493-500 (1958)], porcine liver [ibid., 236, 525-530 (1961)], yeast [ibid., 247, 4794-4802 (1972)] and E. Coli [ibid., 236, 1494-1497 (1961) and 242, 385-392 (1967)].
These NAD synthetases are classified as NAD synthetase (EC 6.3.1.5) which catalyzes the reaction: ##STR4## and NAD synthetase (EC 6.3.5.1.) which catalyzes the reaction: ##STR5##
These NAD synthetases utilize NH.sub.3 and an amide of L-Gln as a substrate, and are differentiatd by the inhibitory action of azaserine.
Km-values of NAD synthetase (EC 6.3.1.5) are reported as 6.5.times.10.sup.-5 M (NH.sub.4 +) and 1.6.times.10.sup.-2 M (L-Gln) [J. Biol. Chem., 1494-1497, (1961), ibid., 242, 385-392 (1967)] and Km-values of NAD synthetase (EC 6.3.5.1) are reported as 6.4.times.10.sup.-8 M (NH.sub.4 +) and 5.times.10.sup.-8 M (L-Gln) [J. Biol. Chem., 247, 4794-4802 (1972), ibid. 233, 493-500 (1958)].
An assay method for NAD synthetase has been reported, in which the generated NAD is reduced by alcohol dehydrogenase (EC 1.1.1) and the absorbency of the generated reduced NAD (hereinafter designated NADH) is spectrophotometrically measured at 340 nm, or the generated NAD is measured by fluorometry.
The above method assays a component in a specimen, selected from ATP, deamide-NAD and an amide donor, and comprises, as a main reaction step, incubating the specimen containing ATP, deamide-NAD or an amide donor such as NH.sub.3, L-glutamine or L-asparagine, with known NAD synthetase (EC 6.3.1.5 and EC 6.3.5.1) in the presence of ATP, deamide-NAD, an amide donor and Mg++ to generate NAD, and further comprises, as a side reaction, performing coenzyme cycling reaction by combining the oxidation-reduction reaction system of coenzyme NAD with the oxidation-reduction reaction system of coenzyme reduced NAD, whereafter a consumed or generated component in the said cycling reaction is measured to effect the assay (Japan Unexam. Pat. Publ. No. 59-198995).
As explained, known NAD synthetases utilize an amide donor substrate of L-glutamine, whereas the present NAD synthetase, which catalyzes at least the reaction (a) hereinbelow in the presence of Mg++ or Mn++ ion, and does not catalyze the reaction (b) hereinbelow in the presence of Mg++ ion, utilizes ammonia including ammonium ion as a substrate and does not utilize at least glutamine and asparagine as a substrate, is heretofore unknown. ##STR6## Also, the known NAD synthetases have substrate specificity on L-glutamine, and so NH.sub.3 cannot be measured using these enzymes in the presence of L-glutamine.